Clinical evaluation of a multitarget fecal immunochemical test‐sDNA test for colorectal cancer screening in a high‐risk population: a prospective, multicenter clinical study

Abstract Colorectal cancer (CRC) is a major malignancy threatening the health of people in China and screening could be effective for preventing the occurrence and reducing the mortality of CRC. We conducted a multicenter, prospective clinical study which recruited 4,245 high‐risk CRC individuals defined as having positive risk‐adapted scores or fecal immunochemical test (FIT) results, to evaluate the clinical performance of the multitarget fecal immunochemical and stool DNA (FIT‐sDNA) test for CRC screening. Each participant was asked to provide a stool sample prior to bowel preparation, and FIT‐sDNA test and FIT were performed independently of colonoscopy. We found that 186 (4.4%) were confirmed to have CRC, and 375 (8.8%) had advanced precancerous neoplasia among the high CRC risk individuals. The sensitivity of detecting CRC for FIT‐sDNA test was 91.9% (95% CI, 86.8–95.3), compared with 62.4% (95% CI, 54.9–69.3) for FIT (P < 0.001). The sensitivity for detecting advanced precancerous neoplasia was 63.5% (95% CI, 58.3–68.3) for FIT‐sDNA test, compared with 30.9% (95% CI, 26.3–35.6) for FIT (P < 0.001). Multitarget FIT‐sDNA test detected more colorectal advanced neoplasia than FIT. Overall, these findings indicated that in areas with limited colonoscopy resources, FIT‐sDNA test could be a promising further risk triaging modality to select patients for colonoscopy in CRC screening.


INTRODUCTION
Colorectal cancer (CRC) is ranked second in incidence and fourth in mortality among all cancers in China, 1 and accumulating evidence suggests that screening is effective for preventing the occurrence and reducing the mortality of CRC. [1][2][3][4][5][6][7] However, the population between 40 and 74 years old is over 600 million in China, which far exceeds the reported annual colonoscopy capability of 5.8 million, 8 making it unfeasible to apply direct colonoscopy for CRC screening. Fecal immunochemical test (FIT) and/or risk-adapted scoring such as high-risk factor questionnaire (HRFQ), 4,9,10 Asia-Pacific Colorectal Screening (APCS) score, 11,12 and modified APCS score have been used for preliminary CRC screening. [13][14][15] Individuals who have positive risk-adapted scores or FIT results are defined as high-risk CRC individuals and are referred to follow-up colonoscopy. This two-stage screening strategy has been performed for decades but has achieved limited success due to high preliminary screening positive rate, low CRC/adenoma detection rate, and poor colonoscopy compliance. 16,17 Therefore, a new test method for further risk triage in individuals who are identified as high-risk of CRC by preliminary screening is urgently needed. Individuals with positive risk-adapted score or a FIT result may complete an additional test to improve the screening effectiveness.
The multitarget fecal immunochemical and stool DNA (FIT-sDNA) test is a novel screening method recommended by United States (US) guidelines for CRC screening in average-risk populations. 6,18 This method detects cancer-related genetic and epigenetic alterations contained in the exfoliated colonic epithelial cells, and it is combined with a specific hemoglobin (Hb) test to derive a final composite score using a logistic-regression algorithm. A large-scale, prospective clinical study in an average-risk US population has demonstrated that the FIT-sDNA test has higher sensitivity in detecting CRC and advanced precancerous neoplasia than FIT. 19 To date, however, there is no large-scale prospective study to evaluate the clinical performance of the FIT-sDNA test for CRC screening for individuals with high-risk profiles.
ColoClear R is a newly developed FIT-sDNA test that consists of molecular assays for KRAS mutation, aberrant methylation of the BMP3 promoter region, and aberrant methylation of the NDRG4 promoter region as well as a hemoglobin test. The methylation target sites were based on Asian population data, and the stool-processing method and logistic-regression algorithm were further optimized to improve the sensitivity.
In the present study, we report the outcome of the prospective cohort from the multicenter clinical trial that targeted high-risk CRC individuals identified by FIT and/or HRFQ. The primary objective was to assess the F I G U R E 1 Study flow chart. Among the 4,245 evaluable participants, 186 participants were diagnosed with CRC (4.4% prevalence), and 375 participants were found to have advanced precancerous neoplasia (8.8% prevalence). Despite the fact that 55.7% of the overall participants were females, 104 (55.9%) participants who had CRC and 241 (64.3%) participants who had advanced precancerous neoplasia were males (Table 1). Among the participants with confirmed CRC, 85 participants had early-stage CRC (Stages I to II), and 140 participants had CRC Stage I to III. CRC was found more frequently in the distal section of the colon than the proximal section (154 vs. 32). Details of the clinical findings are shown in Table S1 (in the Appendix, Supporting information). CRC, colorectal cancer. clinical performance of the FIT-sDNA test for detecting CRC in high-risk individuals, and the secondary objective was to investigate whether the FIT-sDNA test is superior to the conventional FIT in detecting advanced precancerous neoplasia. Our results provide a promising screening modality for further risk triage in high-risk CRC individuals from preliminary CRC screening.

Study population
Between September 2018 and November 2019, a total of 5,241 participants were enrolled from eight tertiary-level hospitals, and 4,245 (80.1%) participants were fully evaluated for the final analysis ( Figure 1 Figure 2A). There was no significant difference in sensitivity according to participants' CRC tumor node metastasis (TNM) stage or tumor location (Figure 2A,B).

The sensitivity of the fecal immunochemical test was inferior to the FIT-sDNA test
The FIT identified 116 out of 181 participants with confirmed CRC with a sensitivity of 62.4% (95% CI, 54.9-69.3; Table 2), which was lower than that of the FIT-sDNA test (P < 0.001). The sensitivities of the FIT for Stages I and II CRC were 68.8% and 77.3%, respectively ( Figure 2A). Compared to the FIT-sDNA test, the sensitivity of the FIT was TA B L E 3 Sensitivity and specificity of the multitarget FIT-sDNA test and FIT for the most advanced findings from colonoscopies in different age groups. Age significantly lower for CRC TNM Stages I-III (Figure 2A), as well as for CRC located proximal or distal of the colon ( Figure 2B). The FIT detected 116 out of 375 participants who had advanced precancerous neoplasia with a sensitivity of 30.9% (95% CI, 26.3-35.6; Table 2). Compared to the FIT-sDNA test, the FIT had a lower sensitivity for detecting advanced precancerous neoplasia. The sensitivity of the FIT for detecting adenomas with high-grade intraepithelial neoplasia was higher than that for detecting adenomas with low-grade intraepithelial neoplasia, but the difference did not reach statistical significance (P = 0.062 for the comparison between high-and low-grade intraepithelial neoplasia; Figure 2C). The sensitivity of the FIT for detecting traditional serrated adenoma (TSA) and sessile serrated adenoma/polyp (SSA/P) was 25% compared to 62.5% of the FIT-sDNA test. For lesions larger than 2 cm, the sensitivity was significantly increased ( Figure 2D). In addition, the sensitivities of the FIT for detecting advanced precancerous neoplasia among participants aged 40 to 49, 50 to 59, 60 to 69, and 70 to 74 years were 29.9% (95% CI, 20.  9) in the 50-to 59-year-old group, 92.8% (95% CI, 90.9-94.3) in the 60-to 69-year-old group, and 92.5% (95% CI, 87.0-95.9) in the 70-to 79-year-old group (Table 3). In addition, the specificities of the FIT in the history of positive fecal occult blood test (FOBT) subgroup, family history of CRC subgroup, clinical symptoms or conditions subgroup, and mixed indications subgroup were 88.8% (95% CI, 82.8-92.9), 97.6% (95% CI, 95.4-98.9), 94.8% (95% CI, 94.0-95.6), and 92.2% (95% CI, 80.3-97.5, Table S3). Overall, the specificities of the FIT were superior to those of the FIT-sDNA test (P < 0.001).

The FIT-sDNA test exhibited the important performance value in CRC screening
To evaluate the performance of the FIT-sDNA test for CRC screening, the number of tests that needed to detect one CRC and one advanced precancerous neoplasia in the study population was calculated in Table S2. To detect TA B L E 4 Colonoscopy finding for colorectal cancer undergoing screening with colonoscopy, multitarget FIT-sDNA test, and FIT in study population.  [16][17][18][19][20], and 37 (95% CI, 30-43), respectively. These data suggested that the performance of the FIT-sDNA test in detecting CRC and advanced precancerous neoplasia was superior to the FIT in the high-risk population. Table 4 showed the detailed data analysis of FIT-sDNA test and FIT among groups with different colonoscopy findings, and the positive prediction value (PPV) and negative prediction value (NPV) were shown in Table 5 (Table 5). Compared to the FIT-sDNA test, the FIT had a slightly higher PPV but lower NPV for advanced colorectal neoplasia.
Using the risk triage strategy in the high-risk population, individuals with positive test results would be referred to immediate follow-up colonoscopy. In a hypothetical condition that the study population adopts this risk triage strategy, 885 (20.8%) would have positive FIT-sDNA test results and subjected to follow-up colonoscopy. Moreover, 409 would be considered true positives, in which advanced colorectal neoplasia is defined as "true finding." In the 3,360 negative test results, 15 CRC and 137 advanced precancerous neoplasia would have been missed and thus defined as false negatives (Table 4). For comparison, 421 (9.9%) would have positive FIT results with 232 true positives, while 70 CRC and 259 advanced precancerous neoplasia cases would be considered false negatives. Further risk triage with the FIT-sDNA test would require 464 more colonoscopies than the FIT group, but the FIT-sDNA test would have detected 177 more advanced colorectal neoplasia cases. The true positive rate for the additional colonoscopy subgroup would be 38.1%, which is 2.8 times the prevalence rate. Overall, these results indicated the important performance value of the FIT-sDNA test in further risk stratification of CRC screening.

DISCUSSION
The present study is the first prospective multicenter clinical study to evaluate the performance of the FIT-sDNA test and conventional FIT in individuals identified as high-risk for CRC by a well-established risk assessment score. The sensitivity of the FIT-sDNA test for detecting advanced colorectal neoplasia was superior to that of the FIT, especially in detecting advanced precancerous neoplasia (62.4% vs. 30.9%). Correspondingly, the FIT-sDNA test had significantly higher NPVs than the FIT in both CRC and advanced precancerous neoplasia. For further risk triage in the high-risk population, sensitivity is the most important factor due to the higher incidence of colorectal neoplasia compared to the average risk population as the priority is to identify more cases and avoid underdiagnosis. In the present study population, the probability of having CRC and advanced colorectal neoplasia with a negative FIT-sDNA test was 0.4% and 4.5% compared to 1.8% and 8.6% with negative FIT results, respectively. Patients with a negative FIT-sDNA test are much less likely to have missed CRC or advanced colorectal neoplasia than those with negative FIT results. Specificity is also important as it determines the number of individuals that may be referred to unnecessary colonoscopies. Currently, the FIT-sDNA test is recommended by national guidelines for screening the average-risk population, but it has a potential negative impact with a relatively high false positive rate in the target population. 18 In the present study, the specificity of the FIT-sDNA test (87.1%-90.3%) was inferior to that of the FIT (94.9%-97.2%). A positive FIT-sDNA test resulted in an increased probability of having CRC from 4.4% to 19.3% compared to 27.6% for the FIT. The probability of having an advanced precancerous lesion with a positive FIT-sDNA test increased from 8.8% to 26.9% compared to 27.6% for a positive FIT result. The FIT-sDNA test produced 464 more positive results than the FIT, and it detected 55 more CRC and 122 more advanced precancerous lesions. The detection rate of advanced colorectal neoplasia among the extra FIT-sDNA test positive results was 38.1%, which was 2.8 times the prevalence rate (13.2%) in the study population. These data suggested that although the FIT-sDNA test produces more positive results than the FIT, it still minimizes the risk of underdiagno-sis and increases the detection rate in a high CRC risk population.
As previously mentioned, the major challenge of preliminary screening using a risk adopted score or the FIT in a limited colonoscopy resource country with a large population are the high positive rate and low detection rate. A recent national wide screening program identified 13% of 1.3 million participants as high-risk CRC individuals by preliminary screening, but the colonoscopy compliance is only 15%. 16 Several studies performed repeated FIT that aims to improve the detection rate and effectiveness but with limited success. 20,21 A recent Chinese expert consensus suggested the potential benefit of the additional FIT-sDNA test for individuals who are identified as high CRC risk by preliminary screening in a limited colonoscopy resource area. 22 This is the first study to demonstrate that comparing with FIT, the FIT-sDNA test is a promising further risk triage modality for individuals who are identified as high CRC risk by preliminary screening. Although an additional FIT may lead to increased detection as well, the FIT-sDNA test is superior to FIT for individuals identified by preliminary screening due to the following reasons. First, FIT-sDNA had a higher sensitivity and detected significant more CRC and advanced precancerous neoplasia than FIT. Noticeably, due to the high incidence of colorectal advanced neoplasia among these individuals, one test with higher sensitivity and NPV is more important and fits the situation better to avoid the risk of miss diagnosis. Second, the colonoscopy compliance was higher for the FIT-sDNA test than FIT. For average-risk population screening, individuals were more likely to prefer the FIT-sDNA test over FIT and the colonoscopy compliance after a positive FIT-sDNA test result has been reported to be higher than after a positive FIT. 23,24 Therefore, for individuals who have a positive risk-adapted score or an FIT result in a limited colonoscopy resource area, an additional FIT-sDNA test can be used to select patients for an urgent follow-up colonoscopy or provided to individuals who refused colonoscopy to increase the colonoscopy compliance.
The present study had several strengths and limitations. This large-scale prospective clinical evaluation demonstrated the superior clinical performance of the FIT-sDNA test compared to the FIT in individuals identified as high CRC risk. Different to prior studies, 16 we targeted individuals who are at high risk for CRC selected by strict inclusion criteria. In the present study, 186 (4.4%) participants were diagnosed with CRC, and 375 (8.8%) participants were diagnosed with advanced precancerous neoplasia. The prevalence of CRC and advanced precancerous neoplasia was comparable to other studies that targeted individuals with a positive FIT or riskadopted score. 8,20,21 However, limitations include that test intervals and cost-effectiveness of the FIT-sDNA test were not analyzed in this study which only focused on the clinical performance of the FIT-sDNA test among the high CRC risk individuals. Although the sensitivity of the FIT-sDNA test was superior to the FIT in the present study, some modeling studies suggested that screening with the FIT-sDNA test every 3 years might not provide a favorable balance of benefits and harms compared with FIT once a year. 6 However, we need to do more longitude follow-up studies with larger sample size and collect more information on labor cost and exact colonoscopy resources to analyze test intervals and cost-effectiveness of the FIT-sDNA test. Second, since each participant was required to provide a stool sample for the FIT-sDNA test in our study, accurate data on the high CRC risk individuals' compliance to the FIT-sDNA test and factors associated with patient preferences were not available. Further real-world studies for evaluation of compliance are warranted. In a word, the exact role of the FIT-sDNA test in CRC screening requires additional investigation beyond the scope of the present study.
In conclusion, the FIT-sDNA test demonstrated significantly higher sensitivities and NPVs for both CRC and advanced precancerous lesions compared to the FIT alone, indicating its potential role for further risk stratification after preliminary screening in CRC screening. The study was designed by the authors and Beijing Mingze Technology, a contract research organization (CRO), provided data collection and monitoring service. A third-party statistician analyzed the data with the leading principal investigator.

Study population
The study population was comprised of participants who were identified as high-risk for CRC by HRFQ which has been widely used as the primary screening method in China and recommended by the Chinese expert consensus on CRC screening and China guidelines for the screening, early detection, and early treatment of CRC. 10,15,22,25 Experienced doctors interviewed participants to collect information about their exposure to CRC risk factors using HRFQ and all data were recorded in case report forms. Briefly, a high-risk population was defined as individuals aged 40 and 74 years and who had at least one of the following risk factors 4,22,[25][26][27] : (1)

Clinical procedures
After providing written informed consent, all eligible participants were required to provide a stool sample for the FIT-sDNA test and FIT prior to bowel preparation and undergo a colonoscopy in the same hospital within 90 days.
Endoscopists followed the guidelines for screening, endoscopic diagnosis, and treatment of early CRC in China. 28 All colonoscopies were performed by experienced endoscopists without knowing the results of either stool test. Abnormal findings during the colonoscopy were sent for pathology examination. Colonoscopy and tissue biopsy pathology results were used as the gold standard in the present study, and the results were reviewed by the pathologist and investigator at each hospital. If two or more colorectal lesions were diagnosed, only the most advanced lesion and its location were used for classification. The adenoma detection rate was 25% in the present study.

Laboratory procedures
Stool specimens were collected and transferred to the medical laboratory at each hospital. Laboratory technicians were blinded to patient identity, clinical history, and the comparator FIT result of the participants. The FIT-sDNA test (ColoClear R , New Horizon Health Technology, China) was performed according to the manufacturer's instructions, and the details of the stool collection and processing are shown in the Supporting information. Briefly, the stool specimen was pretreated with the sample preparation kit provided by the manufacturer, and the integrated FIT test was performed on the same day of stool specimen collection. The DNA extraction and polymerase chain reaction (PCR) analysis were performed within 7 days of the pretreatment. Alternatively, the stool specimen was frozen at −80 • C after finishing the integrated FIT test for subsequent DNA extraction and PCR analysis.
The FIT-sDNA test is a newly developed assay that consists of a qPCR assay for KRAS mutation, aberrant methylation of the BMP3 promoter region, aberrant methylation of the NDRG4 promoter region, β-Actin (ACTB), and B2M (reference gene for DNA quantity) as well as a hemoglobin immunoassay. A logistic-regression algorithm incorporating all the above parameters was used to compute a single composite score with values of 165 or above considered to be positive (Table S4). The detailed information for specimen flow and approach to extraction and analysis of DNA and hemoglobin was shown in Figure S2.
The comparator FIT (FOBT kit (LFD) approved by NMPA, Kangzhu Ltd., China) was performed according to the manufacturer's instructions using the same stool specimen for the FIT-sDNA test. Laboratory technicians were blinded to identify the patient, clinical history, and the FIT-sDNA test results. A red line appeared in the test zone of the FIT kit if the sample concentration exceeded 100 ng Hb/mL.

Outcome
The primary outcome was the sensitivity of the FIT-sDNA test in detecting CRC and the specificity for detecting nonadvanced adenomas or negative colonoscopy findings. The secondary outcome was the sensitivity of the FIT-sDNA test in detecting advanced precancerous neoplasia. The CRC stage was determined according to the 8th edition of Cancer Staging Manual from American Joint Committee on Cancer (AJCC). The definition of advanced adenomas was adenomas with either high-grade intraepithelial neoplasia, villous elements, or measuring ≥1 cm. Advanced precancerous neoplasia was defined as advanced adenomas or traditional sessile adenomas and sessile serrated adenomas/polyps ≥1 cm.

Statistical analysis
The sample size calculation was based on the hypothesis that the sensitivity of the FIT-sDNA test in detecting CRC is no less than 65% and that the specificity of the FIT-sDNA test is no less than 75%. According to the data of the preclinical study submitted to NMPA (conducted by Prof. Shu Zheng and New Horizon Health Technology), the expected sensitivity in detecting CRC was set as 80%. At a two-sided type I error rate of 0.05 and power of 80%, the minimum number required for CRC and patients with nonadvanced adenomas or negative colonoscopy findings was 72 and 563, respectively. The secondary hypothesis was that the sensitivity of the FIT-sDNA test in detecting advanced precancerous neoplasia is superior to the FIT.
To test the secondary hypothesis, the expected sensitivities of the FIT-sDNA test and comparator FIT in detecting advanced precancerous neoplasia were 40% and 25%, respectively. To demonstrate superiority, the required minimal number of valid patients with advanced precancerous neoplasia was 152 with a power of 80% and type I error rate of 0.05. Considering the dropout and loss rate during the clinical study, the required sample sizes for patients with CRC and advanced precancerous neoplasia were 90 and 180, respectively. For patients with nonadvanced adenomas or negative colonoscopy findings, the required sample size was 700. In the present study, we found 186 participants with CRC, 375 participants with advanced precancerous neoplasia, and 3,684 participants with nonadvanced adenomas or negative colonoscopy findings in the final analysis, therefore fulfilling the required sample size for testing the research hypotheses. Sensitivity and specificity with 95% CIs were used to assess the clinical performance for detecting advanced colorectal neoplasia. The McNemar paired-comparison test was used to compare the outcome difference between groups or tests. All analyses were performed using SPSS 22 (International Business Machines Corporation, New York City, the United States of America).

A U T H O R C O N T R I B U T I O N S
Ke-Feng Ding, Yi-You Chen, Ning Lv, Shu Zheng, and Yan-Hong Gu conceived and designed the study. Ye-Ting Hu, Xiao-Feng Chen, Chun-Bao Zhai, Gang Liu, Zhi-Guo Xiong, Zi-Qiang Wang, San-Jun Cai, Wen-Cai Li, Cai-Hua Wang, and Zhi-Hua Tao contributed to the recruitment of participants. Xiang-Xing Kong, Qian Xiao, Guo-Zhong Jiang, Li-Yun Niu, Jian-Long Men, Qing Wang, Shao-Zhong Wei, Jun-Jie Hu, Ting-Han Yang, and Jun-Jie Peng led the data collection. Ye-Ting Hu, Xiao-Feng Chen, and Xiao-Tian Yu contributed to the data analysis and data interpretation. Ye-Ting Hu, Xiao-Tian Yu, and Ke-Feng Ding drafted the manuscript. All authors provided critical review and final approval of the manuscript. The corresponding author attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted.

A C K N O W L E D G M E N T S
We sincerely thank doctors from the eight hospitals for the recruitment of participants, New Horizon Health Technology for providing reagents and Beijing Mingze Technology for providing data collection and monitoring service.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
Xiao-Tian Yu is the Medical Director, Ning Lv is the Chief Technology Officer, and Yi-You Chen is the Chief Scientist at Hangzhou New Horizon Health Technology Company. The other authors declare no conflicts of interest.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The clinical data generated in this study are available within the article and its Supporting information. Additional materials and methods are included in Supporting information. Other detailed data in this study are available upon reasonable request from the corresponding author.

E T H I C S S TAT E M E N T
The Clinical Trials registration number of the study was NCT04287335. The study was carried out on the basis of the guideline of the Declaration of Helsinki and was approved by the Ethics Committees of the Second Affil- O R C I D Qian Xiao https://orcid.org/0000-0002-0120-2371